Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity and functional diversity, playing a key role in developmental decisions, gene expression regulation and, when aberrant, in human disease onset. Importantly, over 1000 splicing variants have been associated with the cancer phenotype. Although the functional analysis of these variants and their possible role in cancer is still in its infancy, their potential importance as cancer biomarkers/therapeutic targets has garnered much interest. We now present a substantial body of preliminary data showing that caffeine, a highly consumed stimulant in the human diet, induces alternative splicing of a large subset of cancer-related genes including the tumor suppressor KLF6. Using KLF6 as a prototype, we have shown that this induction is rapid and reversible and occurs at the level of splicing rather than differential stability. We also demonstrate that the four classic caffeine signaling pathways have little if any role in caffeine-induced alternative splicing, indicating that a novel molecular mechanism is operative. Importantly, since the previous submission we have found that alternative splicing of KLF6 is regulated by the SR protein SC35, and that this protein is induced by caffeine. We now hypothesize that caffeine regulates alternative splicing, at least in part, by the induction of SC35, and may mimic an endogenous pathway aberrantly activated in cancer cells. To further dissect the impact of caffeine on the splicing of cancer-related genes, we now propose to: 1) Investigate the role of SC35 in caffeine-mediated alternative splicing, using KLF6 as a prototype;2) Investigate the interaction of splicing factors with KLF6 ESS-1 and ISE-1 in vitro and in vivo, and interrogate a model for regulation of KLF6 splicing by SC35: 3) Determine the mechanism by which caffeine elevates SC35 and 4) Determine whether caffeine induces "cancer-specific" splice variants in normal tissues in vivo. Our observation that caffeine can regulate alternative splicing of a variety of genes involved in the cancer phenotype is both highly significant and timely. We expect that these studies will result in the identification of elements/factors/pathways that are common to the splicing regulation of this cancer gene subset. We also expect to determine whether high caffeine consumption can induce cancer-related splice variants in normal cells in vivo, resulting in transient expression that may confound both biomarker analysis and targeted therapy.